Single‐Molecule Studies of HIV‐1 Protease Catalysis Enabled by Chemical Protein Synthesis

Abstract

AbstractSingle‐molecule fluorescence studies of the proteolytic activity of the enzyme HIV‐1 protease were performed using FRET‐pair dye labeled peptide substrates and substrate‐derived inhibitors prepared by solid phase peptide synthesis. Chemical protein synthesis was used to prepare homodimeric HIV‐1 protease in soluble form and to prepare a covalent dimer 203 amino acid residue HIV‐1 protease containing a biotin tether at the mid‐point of the synthetic protein molecule. The biotin‐tagged HIV‐1 protease was immobilized on a neutravidin‐coated glass slide. Total internal reflection excitation multiwavelength fluorescence spectroscopy was used to monitor substrate binding and cleavage by the synthetic enzyme molecules. Single‐molecule traces for the dye‐labeled peptide substrate showed distinct binding and cleavage events; the corresponding dye‐labeled peptide inhibitor showed only the binding event. These results constitute strong proof‐of‐principle for the utility of chemical peptide and protein synthesis for single‐molecule studies of enzyme catalysis.